Aggregatibacter actinomycetemcomitans (Aa) and several pathogenic bacteria attach to abiotic surfaces and produce exopolysaccharide that immobilize the bacterial cells on these colonized surfaces. To colonize other virgin surfaces and to overcome starvation associated with overpopulation, cells must detach and disperse from the surfaces. Studies undertaken in the previous funding period have shown that dispersin B (DspB) from Aa is capable of preventing surface attachment of several bacterial species. The use of enzymes that prevent the attachment of bacteria to surfaces through extracellular polymeric substances is an underdeveloped area. We have shown in our studies that DspB depolymerizes exopolysaccharide (PGA) that are made up of -1,6-linked N-acetyl-D-glucosamine and removes biofilms of many Gram negative as well as Gram positive bacterial pathogens. Structure-function studies and other biochemical studies have shown that while DspB is efficient in detachment of PGA-containing biofilms, it also sensitizes the complex biofilms for degradation by additional agents such as DNAse I / II and proteinase K. Our studies have led to the discovery of a second Aa enzyme, PgaB that also possesses the ability to detach biofilms through deacetylation of PGA. PgaB sensitizes DspB for more effective and efficient biofilm detachment. Our long range goal is to develop DspB as a broad-spectrum antibiofilm agent capable of removing complex biofilms in a single step by integrating other enzymes with DspB. Thus, the immediate goals for the current application are to continue to study hybrid enzymes containing DspB and to undertake structure/functional studies on PgaB. Specifically, the following aims will be studied: Specific Aim 1. Design and develop DspB as a broad-spectrum biofilm detachment agent. Specific Aim 2. Identify structural components that contribute to the deacetylase activity of PgaB. Specific Aim 3. Demonstrate that the state of acetylation of PGA contributes to the biofilm formation in Aa.